CDKN2A/p16INK4a Antibody - #AF0228

Fig. 3. Up-regulation of HO-1 attenuated heart aging in vivo. The systemic HO-1 transgenic overexpression aged mice were 16 months. (a) The protein levels of LaminB, p53 and p16 were analyzed by western blot. n = 3. **P b .05. Data are mean ± SEM; one-way ANOVA was used for the statistical analysis. (b) Immunofluorescence staining using anti-p16 and anti-α-actinin antibodies showed the level change of p16. The tissue section thickness is 6 μm. Scale bars represent 100 μm. (c) Immunohistochemistry staining using anti-p16 antibody showed the level change of p16. The tissue section thickness is 6 μm. Scale bars represent 100 μm. (d) Relative levels of SASP containing IL-1, IL-3 and TNF-α were analyzed by qRT-PCR. n = 3 to 4. *P b .01. Data are mean ± SEM; Two-tailed t-test was used for the statistical analysis.

FIGURE 2 PTS reduces reduction of NLRP3 inflammasome and apoptosis in DOX-treated mice. (A) WT C57/BL6J mice were pretreated with PTS (10 mg/kg) and afterwards with DOX (20 mg/kg cumulative dose) for 6 days. Immunochemistry staining of the liver sections with anti-NLRP3 (left, scale bar = 50 μm), the quantification of NLRP3 positive area in each group (right, n = 6); (B) qPCR analyses of NLRP3, IL-1β and IL-18 mRNA levels in each group (n = 6); (C) Western blot analyses of NLRP3, ASC, Caspase-1 p20, IL-1β, and IL-18 proteins and the quantification of the blots in each group (n = 4 per group); (D) Western blot analyses of Cleaved Caspase-3, BAX, and BCL-2 proteins (left) and quantification of the blots in each group (right, n = 4 per group).

Figure 3. Poorly regenerated rat skin shows a decrease in the Fp number and characteristics of aging. (a‒f) Immunofluorescence analysis of (a) LRIG1+ Fps and (d) DLK1+ Frs in the regenerated rat dermis in each group. Quantification of (b) LRIG1+ and (e) DLK1+ cells in the dermis and (c, f) the corresponding mRNA expression; the latter was determined by qPCR and normalized to the expression level in the control group. n = 6. (g) Representative SA-β-gal staining in each group. (h) Representative WB images showing the levels of aging-related proteins such as p16, p21, PPARγ, and TGFβ1 in each group. (i‒l) Bar graph showing mRNA expression of the indicated genes, as determined by qPCR. Expression levels were normalized to the gene expression levels in the control group. n = 6. Bar = 100 μm. Data are presented as the mean ± SEM. One-way ANOVA followed by Tukey’s multiple comparison test was used. ∗P < 0.05, ∗∗P < 0.01. The results are representative of at least three independent experiments. CON denotes normal skin (control group), WELL denotes well-regenerated skin, POOR denotes poorly regenerated skin, and OLD denotes aged skin. Fp, papillary fibroblast; Fr, reticular fibroblast; n.s., nonsignificant; PPARγ, peroxisome proliferator–activated receptor-γ; SA-β-gal, senescence-associated-β-galactosidase; WB, western blot.

Figure 4 SPN attenuated senescence in D-gal-induced mice. (A) Immunohistochemistry of P16, P21, P53 and (B) related statistical analysis (n=5). Data are expressed as the mean ± SD. **P < 0.01 compared with the MOD group.

Figure 5 Detection of p16, IL6, TNF-α, and ROS levels in NPCs. (A) Expression of p16 and IL-6 proteins in NPCs treated with TBHP, with or without PT. (B,C) Expression of p16 and IL-6 proteins relative to β-actin in NPCs treated with TBHP, with or without PT. (D) ROS production in NPCs treated with TBHP, with or without PT. (E) Expression of TNF-α protein in NPCs treated with LPS, with or without PT. (F) Expression of TNF-α protein relative to β-actin in NPCs treated with LPS, with or without PT. Data are the mean ± standard deviation. # p < 0.05, ## p < 0.01 vs. CTL group; * p < 0.05, ** p < 0.01 vs. 100 μM TBHP or 1 μg/mL LPS group.

Figure 2 Evaluation of hippocampal histomorphology in aging mice. (A) H&E and Nissl staining in the hippocampus between 3M and 22M group. The magnifications and scale bars were shown in (A). (B,C) Nissl positive cells and Neurodegeneration rate in the hippocampus were assessed by Nissl staining. Data were represented as mean ± SD and were analyzed by Student t-test. ***P < 0.001. Arrows showed degenerated neurons. (D) IHC of senescence markers P16 and P21 in the hippocampus. The magnifications and scale bars were shown in (D). (E,F) Relative expressions of senescence markers P16 and P21 were calculated by IHC. Data were represented as mean ± SD and were analyzed by Student t-test.

Fig. 6. Senescence characteristics of NC and DOP hJBMMSCs. A. The SA-β-Gal (senescence-associated β-galactosidase) expression in DOP group was significantly higher than that in NC group (Mean ± SD, n = 6). B. The mitochondrial green fluorescence in DOP group was significantly stronger than that in NC group, and the ratio of red/green fluorescence was significantly lower than that of NC group (Mean ± SD, n = 6). C. In the detection of reactive oxygen species, the fluorescence intensity of DOP group was significantly stronger than that of NC group (Mean ± SD, n = 6). D. The expression of senescence related secretory phenotype genes was increased in DOP group (Mean ± SD, n = 3). E. The expression of senescence related proteins in DOP group was increased (Mean ± SD, n = 3). *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3 β-Catenin, cyclin D1, P16, and Ki-67 expression in CP. (A–C) β-Catenin immunostaining score: (A) normal membranous staining in stratified squamous epithelial cells in PCP, (B) moderate and strong nuclear signal in 10%–50% and (C) >50% of neoplastic cells in two ACP cases, respectively. The signal was more represented in cells forming “whorled epithelium” structures. (D–F) Cyclin D1 immunostaining score: low nuclear signal observed in <10% of neoplastic cells (D, a case of PCP); moderate signal observed in 10%–50% of cells (E, a case of PCP); and strong reactivity in >50% of neoplastic cells (F, a case of ACP). (G–I) P16 immunostaining score: (G) signal with low intensity observed in <10% of neoplastic cells in PCP and (H) moderate intensity observed in 10%–50% of neoplastic cells in PCP; and (I) moderate and strong and diffuse (51%–80% of neoplastic cells) immunoreactivity in ACP. (J–L) Ki-67 labeling index: low proliferative index Ki-67 (<5%), and “whorled epithelium” structure (*) (J) extremely low Ki-67 index, (K) high Ki-67 index, and (I) Ki-67 index calculated as 15% in a case of PCP. All pictures were captured at 200× magnification.